Distinct Mechanisms of Pathogenic DJ-1 Mutations in Mitochondrial Quality Control.

The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson's disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein-protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1D149A mutant does not influence mitophagy, but instead impairs Ca2+ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) promotes survival and migration of acute myeloid leukemia cells through CD63/PI3K/Akt/p21 signaling.

We and others have shown that the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), a member of the inflammatory network exerting pleiotropic effects in the bone marrow (BM) microenvironment, regulates the survival and proliferation of different cell types, including normal hematopoietic progenitor cells. Moreover, TIMP-1 has been shown to be involved in cancer progression. However, its role in leukemic microenvironment has not been addressed. Here, we investigated the activity of TIMP-1 on Acute Myelogenous Leukemia (AML) cell functions. First, we found that TIMP-1 levels were increased in the BM plasma of AML patients at diagnosis. In vitro, recombinant human (rh)TIMP-1 promoted the survival and cell cycle S-phase entry of AML cells. These kinetic effects were related to the downregulation of cyclin-dependent kinase inhibitor p21. rhTIMP-1 increases CXCL12-driven migration of leukemic cells through PI3K signaling. Interestingly, activation of CD63 receptor was required for TIMP-1's cytokine/chemokine activity. Of note, rhTIMP-1 stimulation modulated mRNA expression of Hypoxia Inducible Factor (HIF)-1α, downstream of PI3K/Akt activation. We then co-cultured AML cells with normal or leukemic mesenchymal stromal cells (MSCs) to investigate the interaction of TIMP-1 with cellular component(s) of BM microenvironment. Our results showed that the proliferation and migration of leukemic cells were greatly enhanced by rhTIMP-1 in presence of AML-MSCs as compared to normal MSCs. Thus, we demonstrated that TIMP-1 modulates leukemic blasts survival, migration and function via CD63/PI3K/Akt/p21 signaling. As a "bad actor" in a "bad soil", we propose TIMP-1 as a potential novel therapeutic target in leukemic BM microenvironment.

Crucial factors of the inflammatory microenvironment (IL-1ß/TNF-α/TIMP-1) promote the maintenance of the malignant hemopoietic clone of myelofibrosis: an in vitro study.

Along with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of Myelofibrosis (MF). Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1ß, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived circulating CD34+ cells.We found that, regardless mutation status, IL-1ß or TNF-α increases the survival of MF-derived CD34+ cells. In addition, along with stimulation of cell cycle progression to the S-phase, IL-1ß or TNF-α ± TIMP-1 significantly stimulate(s) the in vitro clonogenic ability of CD34+ cells from JAK2V617 mutated patients. Whereas in the JAK2V617F mutated group, the addition of IL-1ß or TNF-α + TIMP-1 decreased the erythroid compartment of the CALR mutated patients. Megakaryocyte progenitors were stimulated by IL-1ß (JAK2V617F mutated patients only) and inhibited by TNF-α. IL-1ß + TNF-α + C-X-C motif chemokine 12 (CXCL12) ± TIMP-1 highly stimulates the in vitro migration of MF-derived CD34+ cells. Interestingly, after migration toward IL-1ß + TNF-α + CXCL12 ± TIMP-1, CD34+ cells from JAK2V617F mutated patients show increased clonogenic ability.Here we demonstrate that the interplay of these inflammatory factors promotes and selects the circulating MF-derived CD34+ cells with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach.

The tissue inhibitor of metalloproteinases 1 increases the clonogenic efficiency of human hematopoietic progenitor cells through CD63/PI3K/Akt signaling.

Initially described as an endogenous inhibitor of proteases, the tissue inhibitor of metalloproteinases 1 (TIMP-1) also displays cytokine-like functions. TIMP-1 is a soluble protein whose levels are increased under inflammatory conditions. We recently found that TIMP-1(-/-) mice have decreased bone marrow (BM) cellularity and that the engraftment capability of TIMP-1(-/-) hematopoietic stem cells (HSCs) is impaired, owing to proliferation defects. Here, we investigated the role of recombinant human TIMP-1 (rhTIMP-1) in human hematopoietic stem/progenitor cells (HSPCs) and elucidated the downstream pathway ignited by rhTIMP-1. We found that rhTIMP-1 affects in vitro cell survival, proliferation, and particularly clonogenic expansion of CD34(+) HSPCs without compromising their short-term engraftment potential after transplantation into immunodeficient mice. These effects are independent on matrix metalloproteinase (MMP) inhibition and rely on TIMP-1's binding to the tetraspanin membrane receptor CD63. Further investigation indicated that rhTIMP-1 stimulation induces phosphatidylinositol 3-kinase (PI3K) recruitment and Akt phosphorylation, both presiding over survival/proliferation pathways in HSPCs. Downstream targets of phosphorylated Akt (pAkt) are also modulated, including the proliferation marker cyclin D1 (CycD1), whose levels are increased upon exposure to rhTIMP-1. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway, suggesting that TIMP-1 might be a key player in the network of proinflammatory factors modulating HSPC functions.

The role of the immunosuppressive microenvironment in acute myeloid leukemia development and treatment.

Functional interplay between acute myeloid leukemia (AML) cells and the bone marrow microenvironment is a distinctive characteristic of this hematological cancer. Indeed, a large body of evidence suggests that proliferation, survival and drug resistance of AML are sustained and modulated by the bone marrow immunosuppressive microenvironment, where both innate and adaptive immune responses are profoundly deregulated. Furthermore, the presence of a number of different immunosuppressive mechanisms results in massive immune deregulation, which causes the eventual escape from natural immune control. Modulating the immune system, as documented by 40 years of stem cell transplantation, may improve survival of AML patients, as the immune system is clearly able to recognize and attack leukemic cells. The understanding of the factors responsible for the escape from immune destruction in AML, which becomes more prominent with disease progression, is necessary for the development of innovative immunotherapeutic treatment modalities in AML.

Hematopoietic stem cell development: an epigenetic journey.

Hematopoietic development and homeostasis are based on hematopoietic stem cells (HSCs), a pool of ancestor cells characterized by the unique combination of self-renewal and multilineage potential. These two opposing forces are finely orchestrated by several regulatory mechanisms, comprising both extrinsic and intrinsic factors. Over the past decades, several studies have contributed to dissect the key role of niche factors, signaling transduction pathways, and transcription factors in HSC development and maintenance. Accumulating evidence, however, suggests that a higher level of intrinsic regulation exists; epigenetic marks, by controlling chromatin accessibility, directly shape HSC developmental cascades, including their emergence during embryonic development, maintenance of self-renewal, lineage commitment, and aging. In addition, aberrant epigenetic marks have been found in several hematological malignancies, consistent with clinical findings that mutations targeting epigenetic regulators promote leukemogenesis. In this review, we will focus on both normal and malignant hematopoiesis, covering recent findings that illuminate the epigenetic life of HSCs.

Extracellular purines promote the differentiation of human bone marrow-derived mesenchymal stem cells to the osteogenic and adipogenic lineages.

Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions, mostly within the processes of tissue damage and repair and flogosis. We previously demonstrated that adenosine 5'-triphosphate (ATP) inhibits the proliferation of human bone marrow-derived mesenchymal stem cells (BM-hMSCs), while stimulating, in vitro and in vivo, their migration. Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes governing adipogenic and osteoblastic (i.e., WNT-pathway-related genes) differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenesis by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator-activated receptor-gamma). In addition, ATP stimulated osteogenic differentiation by promoting mineralization and expression of the osteoblast-related gene RUNX2 (runt-related transcription factor 2). Furthermore, we demonstrated that ATP stimulated adipogenesis via its triphosphate form, while osteogenic differentiation was induced by the nucleoside adenosine, resulting from ATP degradation induced by CD39 and CD73 ectonucleotidases expressed on the MSC membrane. The pharmacological profile of P2 purinergic receptors (P2Rs) suggests that adipogenic differentiation is mainly mediated by the engagement of P2Y1 and P2Y4 receptors, while stimulation of the P1R adenosine-specific subtype A2B is involved in adenosine-induced osteogenic differentiation. Thus, we provide new insights into molecular regulation of MSC differentiation.

Gpr171, a putative P2Y-like receptor, negatively regulates myeloid differentiation in murine hematopoietic progenitors.

Gpr171 is an orphan G-protein-coupled receptor putatively related to the P2Y family of purinergic receptors (P2YRs) for extracellular nucleotides, a group of mediators previously shown to regulate hematopoietic progenitor cells. No information is currently available on the ligand responsible for Gpr171 activation and its biological role remains unknown. We reconstructed Gpr171 phylogenesis in mice and confirmed that Gpr171 is evolutionally related to members of a P2Y gene-cluster localized on mouse chromosome 3. As a first step toward unveiling a role for Gpr171, we investigated its expression profile in murine hematopoietic cells. As opposed to other P2YRs, we found that Gpr171 expression is down-regulated in monocytes and granulocytes, suggesting a negative role in myeloid lineage specification. To test Gpr171 functional role, we next enforced Gpr171 expression in a myeloblastic cell line (32D cells) and in primary Sca-1(+) hematopoietic progenitors, and observed a decreased expression of myeloid markers upon induction of Gpr171, as well as an increased generation of colonies in vitro. Conversely, Gpr171 silencing induced opposite results, diminishing the expression of myeloid markers and the clonogenic potential of 32D cells. In vivo, mice transplanted with hematopoietic progenitor cells overexpressing Gpr171 displayed a significant reduction in the percentage of Mac-1(+)Gr-1(-) cells. As a preliminary step in the investigation of Gpr171 role in murine hematopoiesis, our findings indicate that the orphan receptor Gpr171 negatively regulates myeloid differentiation. Together with phylogenic analyses, our data suggest that Gpr171 may have followed a separate evolutionary pathway as compared to other P2YRs belonging to the same gene cluster.

Less is more: unveiling the functional core of hematopoietic stem cells through knockout mice.

Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cell types. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, Tgf-ß signaling, Pten/Akt signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology.

The sixth sense: hematopoietic stem cells detect danger through purinergic signaling.

Over the past decade, extracellular nucleotides (such as ATP and UTP) have emerged as key immunomodulators. This family of molecules, already known for its key metabolic functions, has been the focus of intense investigation that has unambiguously shown its crucial role as mediators of cell-to-cell communication. More recently, in addition to its involvement in inflammation and immunity, purinergic signaling has also been shown to modulate BM-derived stem cells. Extracellular nucleotides promote proliferation, CXCL12-driven migration, and BM engraftment of hematopoietic progenitor and stem cells. In addition, purinergic signaling acts indirectly on hematopoietic progenitor and stem cells by regulating differentiation and release of proinflammatory cytokines in BM-derived human mesenchymal stromal cells, which are part of the hematopoietic stem cell (HSC) niche. HSC research has recently blended into the field of immunology, as new findings highlighted the role played by immunologic signals (such as IFN-α, IFN-γ, or TNF-α) in the regulation of the HSC compartment. In this review, we summarize recent reports unveiling a previously unsuspected ability of HSCs to integrate inflammatory signals released by immune and stromal cells, with particular emphasis on the dual role of extracellular nucleotides as mediators of both immunologic responses and BM stem cell functions.

Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice.

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.

CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway.

The regulatory mechanisms governing the cell cycle progression of hematopoietic stem cells (HSCs) are well characterized, but those responsible for the return of proliferating HSCs to a quiescent state remain largely unknown. Here, we present evidence that CD81, a tetraspanin molecule acutely responsive to proliferative stress, is essential for the maintenance of long-term repopulating HSCs. Cd81(-/-) HSCs showed a marked engraftment defect when transplanted into secondary recipient mice and a significantly delayed return to quiescence when stimulated to proliferate with 5-fluorouracil (5FU). In addition, we found that CD81 proteins form a polarized patch when HSCs are returning to quiescence. Thus, we propose that the spatial distribution of CD81 during the HSC recovery phase drives proliferative HSC to quiescence, and is important to preserve the self-renewal properties. Here, we show that lack of CD81 leads to loss of HSC self-renewal, and the clustering of CD81 on HSC membrane results in deactivation of Akt, which subsequently leads to nuclear translocation of FoxO1a. Thus, CD81 functions as part of a previously undefined mechanism that prohibits excessive proliferation of HSCs exposed to environmental stress.

Hematopoietic stem cell characterization and isolation.

Hematopoietic stem cells (HSCs) are defined by the capabilities of multi-lineage differentiation and long-term self-renewal. Both these characteristics contribute to maintain the homeostasis of the system and allow the restoration of hematopoiesis after insults, such as infections or therapeutic ablation. Reconstitution after lethal irradiation strictly depends on a third, fundamental property of HSCs: the capability to migrate under the influence of specific chemokines. Directed by a chemotactic compass, after transplant HSCs find their way to the bone marrow, where they eventually home and engraft. HSCs represent a rare population that primarily resides in the bone marrow with an estimated frequency of 0.01% of total nucleated cells. Separating HSCs from differentiated cells that reside in the bone marrow has been the focus of intense investigation for years. In this chapter, we will describe in detail the strategy routinely used by our laboratory to purify murine HSCs, by exploiting their antigenic phenotype (KSL), combined with the physiological capability to efficiently efflux the vital dye Hoechst 33342, generating the so-called Side Population, or SP.

TIMP-1 deficiency subverts cell-cycle dynamics in murine long-term HSCs.

In addition to the well-recognized role in extracellular matrix remodeling, the tissue inhibitor of metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biologic functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of HSCs, whose biologic behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1(-/-) mice have decreased BM cellularity and, consistent with this finding, TIMP-1(-/-) HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1(-/-) stem cells appears distorted, with a dysregulation at the level of the G(1) phase. TIMP-1(-/-) HSCs also display increased levels of p57, p21, and p53, suggesting that TIMP-1 could be intrinsically involved in the regulation of HSC cycling dynamics. Of note, TIMP-1(-/-) HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G(1)/S transition. Our findings establish a role for TIMP-1 in regulating HSC function, suggesting a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu.

The expansion of T-cells and hematopoietic progenitors as a result of overexpression of the lymphoblastic leukemia gene, Lyl1 can support leukemia formation.

This study investigates the function of the lymphoblastic leukemia gene, Lyl1 in the hematopoietic system and its oncogenic potential in the development of leukemia. Overexpression of Lyl1 in mouse bone marrow cells caused T-cell increase in the peripheral blood and expansion of the hematopoietic progenitors in culture and in the bone marrow. These observations were the result of increased proliferation and suppressed apoptosis of the progenitor cells caused by the Lyl1-overexpression. Our studies present substantial evidence supporting the secondary, pro-leukemic effect of Lyl1 in early hematopoietic progenitors with the potential to cause expansion of malignant cells with a stem/early progenitor-like phenotype.

Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12 and increases the homing capacity and production of proinflammatory cytokines.

OBJECTIVE: Extracellular adenosine triphosphate (ATP) is a well-recognized mediator of cell-to-cell communication. Here we show ATP effects on bone marrow (BM)-derived human mesenchymal stem cell (hMSCs) functions. MATERIALS AND METHODS: ATP-induced modification of hMSCs gene expression profile was assessed by Affymetrix technology. Clonogenic and migration assays in vitro, as well as xenotransplant experiments in vivo, were performed to evaluate the effects of ATP on hMSCs proliferation and BM homing. Enzyme-linked immunosorbent assays were used to assess hMSCs cytokines production, whereas T-cell cultures demonstrated the immunoregulatory activity of ATP-treated hMSCs. RESULTS: hMSCs were resistant to the cytotoxic effects of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP-stimulated hMSCs underwent a downregulation of genes involved in cell proliferation, whereas those involved in cell migration were strongly upregulated. The inhibitory activity of ATP on hMSCs proliferation was confirmed by assessing clonogenic stromal progenitors. ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, the homing capacity of hMSCs to the BM of immunodeficient mice was significantly increased by pretreatment with ATP. Moreover, ATP increased the production of the proinflammatory cytokines interleukin-2, interferon-γ, and interleukin-12p70, while decreasing the anti-inflammatory cytokine interleukin-10, and this finding was associated with the reduced ability of MSCs to inhibit T-cell proliferation. CONCLUSIONS: Our data show that purinergic signaling modulates hMSCs functions and highlights a role for extracellular nucleotides in hMSCs biology.

Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib.

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin(-)CD34(-)) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34(-) cells are leukemic. CML Lin(-)CD34(-) cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin(-)CD34(-) cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34(+) cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin(-)CD34(-) cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin(-)CD34(-) cells in vitro. Moreover, leukemic CD34(-) cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34(-) leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration.

Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and nonpeptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine triphosphate (ATP) and uridine triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation by migrating CD34+ cells, and increased cell adhesion to fibronectin. In vivo, preincubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Galphai) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP- and CXCL12-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5'-triphosphatase (GTPase) Rac2 and downstream effectors Rho GTPase-activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the homing of HSCs to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Galphai proteins and RhoGTPases.

The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment.

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior.

Extracellular nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo.

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells, little is known about their effect on blood cell progenitor cells. In this study, we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs), and whether activation by their natural ligands, adenosine triphosphate (ATP) and uridine triphosphate (UTP), induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore, stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally, ATP and, to a higher extent, UTP acted as potent early acting growth factors for HSCs, in vitro, because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore, xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions.